Diazonium salt modification of silk polymer

ABSTRACT

A method for modifying silk polymer by coupling a chemical moiety to a tyrosine residue of a silk polymer is described herein for the purpose of altering the physical properties of the silk protein. Thus, silk proteins with desired physical properties can be produced by the methods described herein. These methods are particularly useful when the introduction of cells to a mammal is desired, since modifications to the silk protein affect the physical properties and thus the adhesion, metabolic activity and cell morphology of the desired cells. The silk protein can be modified to produce, or modify, a structure that provides an optimal environment for the desired cells.

CROSS REFERENCE

This application is a continuation under 35 U.S.C. 120, 121, or 365(c)of U.S. patent application Ser. No. 12/192,588 filed Aug. 15, 2008 whichclaims the benefit of priority as applicable under 35 U.S.C. 119(e) ofU.S. Provisional Application Ser. Nos. 61/077,593 filed on Jul. 2, 2008and 61/036,284 filed on Mar. 13, 2008, the contents of each of theseapplications is incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with Government support under The United StatesAir Force Office of Scientific Research—FA9550-07-1-0079; NationalInstitutes of Health—NIH P41 EB002520; and National Institutes of Healthand National Institute of Arthritis and Musculoskeletal and SkinDiseases—NIH F32 AR055029. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to modified silk polymer.

BACKGROUND

A major goal of tissue repair and regeneration is to develop abiological alternative in vitro for producing an implantable structurethat serves as a support and speeds regenerative growth in vivo within adefect area.

In recent years biodegradable polymers such as poly (glycolic acid),poly (L-lactic acid) (PLLA) and their copolymerspoly(L-lactic-co-glycolic acid) (PLGA) have been used as scaffoldmaterials in studies of tissue formation (Sofia S., Functionalizedsilk-based biomaterials for bone formation, J. Biomed. Mater. Res.,54(1):139-148 (2001)). Advantages of these polymers include theirbiocompatibility and degradability. However, PLGA can induceinflammation due to the acid degradation products that result duringhydrolysis (Sofia, S., (2001)). There are also processing difficultieswith polyesters that can lead to inconsistent hydrolysis rates andtissue response profiles. Thus, there is a need for polymeric materialsthat have more controllable features such as hydrophobicity,hydrophilicity, structure, and mechanical strength, while also beingbiocompatible and/or bioresorbable. Biological polymeric materials oftendemonstrate combinations of properties which are unable to be reproducedby synthetic polymeric materials. (Perez-Rigueiro et al. Science, 1998;70: 2439-2447; Hutmacher D. Biomaterials 2000. 21, 2529-2543). Forexample, scaffolds for bone tissue regeneration require high mechanicalstrength and porosity along with biodegradability and biocompatibility.

Silk fibroin isolated from Bombyx mori silkworm cocoons has beenemployed as a matrix material in many tissue engineering applications(Altman G H, et al Biomaterials 2003; 24(3):401-16; Wang Y, et al.Biomaterials 2006; 27(36):6064-82; Kim H J, et al. Macromol Biosci 2007;7(5):643-55; Hofmann S, et al. Biomaterials 2007; 28(6):1152-62; Wang Y,et al Biomaterials 2006; 27(25):4434-42; Meinel L, Bone 2006;39(4):922-31; Hofmann S, et al. Tissue Eng 2006; 12(10):2729-38; AltmanG H, et al. Biomaterials 2002; 23(20):4131-41) due to its mechanicalproperties (Heslot H. Biochimie 1998; 80(1):19-31), biocompatibility(Meinel L, et al. Biomaterials 2005; 26(2):147-55), slow degradationprofile (Horan R L, et al. In vitro degradation of silk fibroin.Biomaterials 2005; 26(17):3385-93), and aqueous processibility (Wang X,et al. J Control Release 2007; 117(3):360-70; Wang X, et al. J ControlRelease 2007; 121(3):190-9; Li C, et al. Biomaterials 2006;27(16):3115-24; Karageorgiou V, et al. J Biomed Mater Res A 2006;78(2):324-34; Wang X, et al. Langmuir 2005; 21(24):11335-41; Kim U J, etal. Biomaterials 2005; 26(15):2775-85; Nazarov R, et al.Biomacromolecules 2004; 5(3):718-26; Kim U J, et al. Biomacromolecules2004; 5(3):786-92; Jin H J, et al. Biomaterials 2004; 25(6):1039-47; JinH J, et al. Biomacromolecules 2002; 3(6):1233-9). The mechanicalproperties of the silk fibroin protein can be attributed to theformation of an extended crystalline β-sheet structure that is composedof recurrent sequences of glycine, alanine and serine amino acids(Heslot, H., (1998); Lotz B, et al. Biochimie 1979; 61(2):205-14; Zhou CZ, et al. Proteins 2001; 44(2):119-22). The extent of β-sheet structureformation can be controlled through physical (Kim U J, et al. (2005);Valluzzi R, et al. Philos Trans R Soc Lond B Biol Sci 2002;357(1418):165-7) or chemical methods (Winkler S, et al. Biochemistry2000; 39(41):12739-46; Matsumoto A, et al. J Phys Chem B 2006;110(43):21630-8; H.-J. Jin J P et al, 2005; 15(8):1241-1247), leading tomaterials with controlled crystallinity and degradation rate. In orderto further enhance robust tissue formation in vitro using silk as ascaffolding material, tailoring the interaction between these scaffoldsand human bone marrow-derived mesenchymal stem cells (hMSCs) isdesirable. Adult hMSCs offer potential for regenerative therapies, asthey are able to differentiate into bone (Kraus K H, Kirker-Head C. VetSurg 2006; 35(3):232-42; Mauney J R, et al. Tissue Eng 2005;11(5-6):787-802), cartilage (Djouad F, et al. Regen Med 2006;1(4):529-37; Magne D, et al. Trends Mol Med 2005; 11(11):519-26), fat(Neumann K, et al. J Cell Biochem 2007; Mauney J R, et al. Biomaterials2005; 26(31):6167-75), muscle (Pittenger M, et al. J MusculoskeletNeuronal Interact 2002; 2(4):309-20), and ligament (Vunjak-Novakovic G,et al. Annu Rev Biomed Eng 2004; 6:131-56) cell lines.

SUMMARY OF THE INVENTION

Disclosed herein are methods for modifying a silk protein for thepurpose of tailoring physical properties of the silk for a desiredapplication, for example a scaffold for human mesenchymal stem cells.More specifically, a chemical moiety is attached to a tyrosine residueof the silk protein by diazonium coupling chemistry, which can alter thehydrophobicity, or conversely the hydrophilicity of the silk protein.Modified silks can be used in a variety of ways, which are known tothose skilled in the art. It is preferred that the modified silkproteins disclosed herein are used to modify an implantable structure(e.g., a medical device) or a tissue culture platform, such that thesilk is flexibly tailored for the desired application. For example, amodified silk can be used for culturing cells or as a bioimplantablescaffold. The biological applications of modified silks is vast andinclude, for example structural supports for bone and tissueregeneration and/or drug delivery.

In one aspect a method for producing a modified silk composition isdescribed, which comprises the steps of contacting a diazonium salt witha silk polymer solution to form a modified silk mixture, and thentransforming the modified silk mixture into an insoluble state to form amodified silk composition. Alternatively, a modified silk compositioncomprises the steps of transforming a silk polymer solution into aninsoluble state, and contacting the insoluble silk polymer with adiazonium salt to form a modified silk composition.

In one embodiment of this aspect and all other aspects disclosed herein,the silk polymer and diazonium salt are mixed to form a substantiallyhomogeneous mixture.

In another embodiment of this aspect and all other aspects disclosedherein, silk polymer contains at least one tyrosine residue and thediazonium salt contains at least one chemical moiety.

In another embodiment of this aspect and all other aspects disclosedherein, contacting involves reacting at least one of the chemicalmoieties of the diazonium salt with the tyrosine residue of the silkpolymer.

In another embodiment of this aspect and all other aspects disclosedherein, a chemical moiety is selected from the group consisting of asulfonic acid group, a carboxylic acid group, an amine group, a ketonegroup, an alkyl group, an alkoxy group, a thiol group, a disulfidegroup, a nitro group, an aromatic group, an ester group, an amide groupand a hydroxyl group. In another embodiment of this aspect and all otheraspects described herein, a chemical moiety is attached to a tyrosineresidue of the silk polymer.

In another embodiment of this aspect and all other aspects disclosedherein, the modified insoluble silk polymer is hydrophilic and inanother embodiment the modified silk polymer is hydrophobic.

In another embodiment of this aspect and all other aspects disclosedherein, the silk polymer further comprises a bioactive agent. In anotherembodiment of this aspect and all other aspects disclosed herein, thebioactive agent comprises small molecules, protein, peptides or nucleicacids.

In another embodiment of this aspect and all other aspects disclosedherein, the bioactive agent is bonded to the chemical moiety of themodified silk protein.

In another embodiment of this aspect and all other aspects disclosedherein, the silk polymer further comprises a mineral. In anotherembodiment of this aspect and all other aspects disclosed herein, themineral is contacted with the silk polymer prior to contact with thediazonium salt or alternatively, the minerals are contacted with thesilk polymer after contact with the diazonium salt. In some embodiments,the mineral is biocompatible with bone or cartilage.

In another embodiment of this aspect and all other aspects disclosedherein, the silk polymer further comprises cells.

In another embodiment of this aspect and all other aspects disclosedherein, the cell is selected from the group consisting of a stem cell, aprimary cell and a cell line.

In another embodiment, the stem cells are induced to differentiate,while in alternative embodiment the stem cells are maintained in anundifferentiated state.

In another aspect, the method for modifying silk comprises the steps offorming the silk into an insoluble state, and contacting a diazoniumsalt with the insoluble silk.

In another aspect a method for modifying a surface is described, whichcomprises the steps of (a) contacting a diazonium salt with a silkfibroin solution to form a modified silk mixture, (b) coating a surfacewith the modified silk mixture, and (c) transforming the modified silkmixture into an insoluble state.

In one embodiment of this aspect and all other aspects disclosed herein,the surface comprises an implantable structure. Alternatively, thesurface comprises a tissue culture platform.

Another aspect disclosed herein is a composition of modified silk, thecomposition comprising silk polymer that has been modified by (a) areaction with a diazonium salt having at least one chemical moiety, and(b) a transformation into the insoluble state.

In one embodiment the composition further comprises a bioactive agent.

In another embodiment the composition of modified silk comprises achemical moiety selected from the group consisting of a sulfonic acidgroup, a carboxylic acid group, an amine group, a ketone group, an alkylgroup, an alkoxy group, a thiol group, a disulfide group, a nitro group,an aromatic group, an ester group, an amide group and a hydroxyl group.

In another embodiment of this aspect and all other aspects describedherein, the silk polymer contains at least one tyrosine residue.

In another embodiment of this aspect and all other aspects describedherein, the chemical moiety is bonded to the tyrosine residue.

In another embodiment of this aspect, the modified silk composition isformed into an insoluble state.

In another embodiment, the insoluble silk polymer is hydrophilic.Alternatively, the insoluble silk polymer is hydrophobic.

In another embodiment of this aspect, the bioactive agent comprisessmall molecules, proteins, polypeptides, or nucleic acids. In anotherembodiment, the bioactive agents are bonded to the chemical moiety of amodified silk.

In one embodiment the silk polymer further comprises a mineral. In analternative embodiment, the mineral is biocompatible with bone orcartilage.

In another embodiment, the silk polymer further comprises cells, whichare selected from the group consisting of a stem cell, a primary celland a cell line. In another embodiment the stem cells are induced todifferentiate. Alternatively, the stem cell is maintained in anundifferentiated state.

In another embodiment of this aspect and all other aspects disclosedherein, the modified silk composition is in a form useful for an opticsapplication.

Also disclosed herein is a kit for modifying silk protein, whichcomprises silk polymer, diazonium salt, and packaging materialstherefor.

DEFINITIONS

As used herein, the term “silk polymer” or “silk fibroin” includessilkworm fibroin and insect or spider silk protein (Lucas et al., Adv.Protein Chem 13: 107-242 (1958). Preferably, fibroin is obtained from asolution containing a dissolved silkworm silk or spider silk. Generally,silk polymer of silk fibroin has been treated to substantially removesericin. The silkworm silk protein is obtained, for example, from Bombyxmori, and the spider silk is obtained, for example, from Nephilaclavipes. In the alternative, silk proteins suitable for use in thepresent invention can be obtained from a solution containing agenetically engineered silk, such as from bacteria, yeast, mammaliancells, transgenic animals or transgenic plants. See, for example, WO97/08315 and U.S. Pat. No. 5,245,012.

As used herein, the term “modified silk” or “modified silk composition”refers to a silk polymer following contact with a diazonium salt. A silkpolymer as described herein, is “modified” by a diazonium couplingreaction, wherein a desired chemical moiety is bonded to a tyrosineresidue in the silk polymer. A “chemical moiety” is a chemical sidegroup that can be used to change the physical properties of a molecule,for example hydrophobicity, hydrophilicity, or gelation time. Somenon-limiting examples of chemical moieties include a sulfonic acidgroup, a carboxylic acid group, an amine group, a ketone group, an alkylgroup, an alkoxy group, a thiol group, a disulfide group, a nitro group,an aromatic group, an ester group, an amide group or a hydroxyl group.In some embodiments, the chemical moiety of a modified silk may alsobind another moiety, for example a bioactive agent such as a drug, or aprotein, wherein the chemical moiety has an additional function as alinker as well as the effect of the chemical moiety on the physicalproperties of a silk protein.

As used herein, the term “diazonium salt” refers to a group of organiccompounds with a structure of R—N₂ ⁺X⁻, wherein R can be any organicresidue (e.g., alkyl or aryl) and X is an inorganic or organic anion(e.g., halogen). A diazonium salt can be formed by the treatment ofaromatic amines (e.g., aniline) with sodium nitrite in the presence of amineral acid and methods for synthesizing diazonium salts are known tothose of skill in the art. See for example WO 2006/014549, WO2004/108633 and WO 2001/025341, which are incorporated herein byreference.

As used herein the term “insoluble state” refers to the formation of, orstate of being in, a substantially amorphous, primarily β-sheetconformation. The term ‘transformed into an insoluble state’ is notintended to reflect polymerization of silk monomers into a silk polymer.Rather, it is intended to reflect the conversion of soluble silk polymerto a water insoluble state. As used herein silk polymer is in an‘insoluble state’ if it can be pelleted by centrifugation or if itcannot be dissolved by immersion in, or rinsing with, water at 37° C. orless.

As used herein, the term “surface” is used to describe the portion ofany structure that can be modified by forming silk into an insolublestate on the exterior or interior portion of the structure. The surfacecan be made of any material, wherein the material is non-toxic to cellsand is therefore capable of sustaining cell viability under appropriateculture conditions for the cell.

As used herein, the term “implantable structure” is generally anystructure that upon implantation does not generate an immune response inthe host organism. Thus, an implantable structure should not forexample, be or contain an irritant, or contain LPS etc. The term“implantable structure” can be used to describe a medical device made toreplace or act as a missing biological structure. In this contextimplants may be placed within the body (internal) or placed outside thebody (external). An implantable structure can also be a drug deliverydevice such as a hydrogel or a subcutaneous implant that for example,can allow sustained or long-term delivery of an agent.

As used herein, the term “tissue culture platform” is used to describeany surface on which an adherent mammalian cell can attach to and/ormaintain cell viability. The “tissue culture platform” can includeculture dishes, plates, wells, slides, discs and coverslips, amongothers. The “tissue culture platform” can also be an engineered scaffoldof desired shape/size.

As used herein, the term “induced to differentiate” refers to achemical/biological treatment, a physical environment or a geneticmodification that is conducive to the formation of terminallydifferentiated cells (e.g., cardiomyocytes or neurons) from pluripotentor multipotent stem cells (e.g., mesenchymal stem cells).Differentiation can be assessed by the appearance of distinct cell-typespecific markers or by the loss of stem cell specific markers. The term“pluripotent” is used to denote cells that are capable of formingterminally differentiated cells of all lineages, whereas the term“multipotent” is used to denote cells that are capable of formingterminally differentiated cells of a particular lineage, for examplehematopoetic cells.

As used herein, the term “form useful for an optics application” is usedto describe the modified silks formed into optical devices such as, forexample diffraction gratings, pattern generators, and lenses. Variousoptical applications for use with the modified silks described hereinare contemplated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. shows a schematic depiction of a diazonium coupling reaction insilk.

FIG. 2. depicts non-limiting examples of aniline derivatives that can beused to modify silk.

FIG. 3. depicts the characterization of an exemplary diazonium saltmodified silk polymer. Examples of (a) a UV/vis spectra of a modifiedsilk polymer; (b) ¹H-NMR spectra, and (c) the percentage of estimatedmodified tyrosine residues are shown.

FIG. 4. shows an example of a UV/vis spectra of modified silks comparedto unmodified silk.

FIG. 5. shows an example ATR-FTIR spectra, which can be used to test forβ-sheet formation.

FIG. 6. depicts one example of metabolic activity of cells grown on anexemplary modified silk.

FIG. 7. shows microscope images of a representative example of cellmorphology on exemplary modified silk polymers.

FIG. 8. shows representative microscope images of live cells grown onexemplary modified silk polymers.

FIG. 9. is a series of graphs that illustrate the use of RT-PCR tomeasure expression levels of exemplary cell adhesion markers in aculture of cells.

FIG. 10. is a series of graphs that illustrate the use of RT-PCR tomeasure expression levels of exemplary differentiation markers in aculture of cells.

Table 1. shows the contact angle of water on representative modifiedsilks.

DETAILED DESCRIPTION OF THE INVENTION

A simple chemical modification method using diazonium coupling chemistrywas developed to tailor the structure and hydrophilicity of silk fibroinprotein and is disclosed herein. The methods disclosed herein can beused to install small molecules with various functional groups includingsulfonic and carboxylic acids, amines, ketones and alkanes, amongothers, for the purpose of tailoring the physical properties of a silkpolymer. Thus, a diazonium salt with a pre-selected side chain can beused to form a silk polymer with desired physical properties such as forexample, altered hydrophilicity/hydrophobicity or gelation time.

Isolation of Silk Polymer

Silk is a well described natural fiber produced by the silkworm, Bombyxmori, which has been used traditionally in the form of threads intextiles for thousands of years. This silk contains a fibrous proteintermed fibroin (both heavy and light chains) that forms the thread core,and glue-like proteins termed sericin that surround the fibroin fibersto cement them together. The fibroin is a highly insoluble proteincontaining up to 90% of the amino acids glycine, alanine and serineleading to β-pleated sheet formation in the fibers (Asakura, et al.,Encyclopedia of Agricultural Science, Arntzen, C. J., Ritter, E. M.Eds.; Academic Press: New York, N.Y., 1994; Vol. 4, pp 1-11).

Silk provides an important set of material options for biomaterials andtissue engineering because of the impressive mechanical properties,biocompatibility and biodegradability (Altman, G. H., et al.,Biomaterials 2003, 24, 401-416; Cappello, J., et al., J. Control.Release 1998, 53, 105-117; Foo, C. W. P., et al., Adv. Drug Deliver.Rev. 2002, 54, 1131-1143; Dinerman, A. A., et al., J. Control. Release2002, 82, 277-287; Megeed, Z., et al., Adv. Drug Deliver. Rev. 2002, 54,1075-1091; Petrini, P., et al., J. Mater. Sci-Mater. M. 2001, 12,849-853; Altman, G. H., et al., Biomaterials 2002, 23, 4131-4141;Panilaitis, B., et al., Biomaterials 2003, 24, 3079-3085). The uniquemechanical properties of reprocessed silk such as fibroin and itsbiocompatibility make the silk fibers especially attractive for use inbiotechnological materials and medical applications. For example,3-dimensional porous silk scaffolds have been described for use intissue engineering (Meinel et al., Ann Biomed Eng. 2004 January;32(1):112-22; Nazarov, R., et al., Biomacromolecules in press). Further,regenerated silk fibroin films have been explored as oxygen- anddrug-permeable membranes, supports for enzyme immobilization, andsubstrates for cell culture (Minoura, N., et al., Polymer 1990, 31,265-269; Chen, J., et al., Minoura, N., Tanioka, A. 1994, 35, 2853-2856;Tsukada, M., et al., Polym. Sci. Part B Polym. Physics 1994, 32,961-968). In addition, silk hydrogels have found numerous applicationsin tissue engineering, as well as in drug delivery (Megeed et al., PharmRes. 2002 July; 19(7):954-9; Dinerman et al., J Control Release. 2002Aug. 21; 82(2-3):277-87).

A method for modifying silk polymer by coupling a chemical moiety to atyrosine residue of a silk polymer is described herein for the purposeof altering the physical properties of the silk protein. Thus, silkproteins with desired physical properties can be produced by the methodsdescribed herein. These methods are particularly useful when theintroduction of cells to a mammal is desired, since modifications to thesilk protein affect the physical properties and thus the adhesion,metabolic activity and cell morphology of the desired cells. The silkprotein can be modified to produce, or coat, a structure, which providesan optimal environment for the culture or delivery of desired cells. Oneembodiment of this method is outlined in Example 1.

All-Aqueous Isolation of Silk Polymer

In some cases it can be advantageous to avoid the use of organicsolvents in the isolation of silk polymer, for example when the silkpolymer is to be combined with living cells. Such solvents can posebiocompatibility problems when the processed materials are exposed tocells in vitro, or in vivo. Further, where it is desired to include abioactive agent e.g., a growth factor or other bioactive molecule in astructure to be implanted, it can be advantageous to use an all-aqueousapproach in order to maintain the activity of the agent.

Organic solvents can also change the properties of fibroin material. Forexample, the immersion of silk fibroin films in organic solvents such asmethanol causes dehydration of the hydrated or swollen structure,leading to crystallization and thus, loss of solubility in water.Further, with respect to tissue engineering scaffolds, the use oforganic solvents can render the silk material to be less degradable.

An all-aqueous approach for preparing silk to be used is described, forexample, in U.S. 20070187862, which is incorporated herein by reference.

Diazonium Salts

Diazonium salts useful for the methods and compositions describedherein, are known to those of skill in the art. A diazonium saltcomprises a group of organic compounds with a structure of R—N₂ ⁺X⁻,wherein R can be any organic residue (e.g., alkyl or aryl) and X is aninorganic or organic anion (e.g., halogen). A diazonium salt can beformed by the treatment of aromatic amines (e.g., aniline) with sodiumnitrite in the presence of a mineral acid and methods for synthesizingdiazonium salts are known to those of skill in the art. See for exampleWO 2006/014549, WO 2004/108633 and WO 2001/025341, which areincorporated herein by reference. The methods for synthesizing diazoniumsalts and the chemistries involved in diazonium coupling are well withinthe ability of one skilled in the art for use with the methods describedherein. Diazonium salts for use herein comprise at least one chemicalmoiety, however it is also contemplated that a diazonium salt comprisesmultiple chemical moieties.

Chemical Modification of Residues

The physical properties and surface chemistry of the silk protein can bemodified by bonding a chemical moiety to a tyrosine residue in the silkprotein and is particularly useful for synthesizing a structure orsurface designed for attaching or growing cells. Biomaterial surfacechemistry is known to influence a variety of cell responses ranging fromchanges in surface adhesion (Sofia S, McCarthy M B, Gronowicz G, KaplanD L. J Biomed Mater Res 2001; 54(1):139-48; Kim H J, Kim U J,Vunjak-Novakovic G, Min B H, Kaplan D L. Biomaterials 2005;26(21):4442-52; Uebersax L, Hagenmuller H, Hofmann S, Gruenblatt E,Muller R, Vunjak-Novakovic G, et al. Tissue Eng 2006; 12(12):3417-29) toactivation of biochemical pathways regulating cellular proliferation,differentiation, and survival (Sofia, S. et al (2001); Kim, H J, et al(2005)1 Uebersax, L, et al (2006); Keselowsky B G, Collard D M, Garcia AJ. Proc Natl Acad Sci USA 2005; 102(17):5953-7). For example, surfacehydrophilicity can affect cell adherence and proliferation (Kim M S,Shin Y N, Cho M H, Kim S H, Kim S K, Cho Y H, et al. Tissue Eng 2007;13(8):2095-103; Grinnell F. Int Rev Cytol 1978; 53:65-144) and regulateexpression of specific cell surface integrins (Keselowsky, B G, e tal(2005); Garcia A J, Boettiger D. Biomaterials 1999; 20(23-24):2427-33;Garcia A J, Gallant N D. Cell Biochem Biophys 2003; 39(1):61-73; GarciaA J. Biomaterials 2005; 26(36):7525-9; Hubbell J A. Biotechnology (N Y)1995; 13(6):565-76; Lim J Y, Taylor A F, Li Z, Vogler E A, Donahue H J.Tissue Eng 2005; 11(1-2):19-29; Keselowsky B G, Collard D M, Garcia A J.Biomaterials 2004; 25(28):5947-54; Grinnell F, Milam M, Srere P A. ArchBiochem Biophys 1972; 153(1):193-8). Substrate surface chemistry hasalso been shown to affect cell differentiation directly (Keselowsky B G,(2005); Lim J Y, et al (2005); Keselowsky B G, et al (2004); Liu X, LimJ Y, Donahue H J, Dhurjati R, Mastro A M, Vogler E A. Biomaterials 2007;28(31):4535-50; Curran J M, Chen R, Hunt J A. Biomaterials 2006;27(27):4783-93, Curran J M, Chen R, Hunt J A. Biomaterials 2005;26(34):7057-67) or by modulating the adsorption of extracellular matrixproteins such as fibronectin, vitronectin and laminin to the substrate(Keselowsky, B G., et al (2005), Keselowsky, B G., et al (2004);Keselowsky B G, Collard D M, Garcia A J. J Biomed Mater Res A 2003;66(2):247-59). These matrix proteins can selectively engage cell surfaceintegrins that are critical for differentiation (Takeuchi Y, Suzawa M,Kikuchi T, Nishida E, Fujita T, Matsumoto T. J Biol Chem 1997;272(46):29309-16; Xiao G, Wang D, Benson M D, Karsenty G, Franceschi RT. J Biol Chem 1998; 273(49):32988-94; Keselowsky B G, Wang L, SchwartzZ, Garcia A J, Boyan B D. J Biomed Mater Res A 2007; 80(3):700-10; KunduA K, Putnam A J. Biochem Biophys Res Commun 2006; 347(1):347-57). Theability to modify the surface chemistry of a biomaterial can also aid inconstruction of a synthetic tissue with physical properties similar tothat of native tissue (Hutmacher D W. Biomaterials 2000;21(24):2529-43). For bone tissue engineering, modification ofbiomaterial scaffolds with charged groups (such as carboxylic acids)that are conducive to mineralization can facilitate pre-mineralizationof the scaffold with hydroxyapatite (Chen J, Chu B, Hsiao B S. J BiomedMater Res A 2006; 79(2):307-17; Goissis G, da Silva Maginador S V, daConceicao Amaro Martins V. Artif Organs 2003; 27(5):437-43; Song J,Malathong V, Bertozzi C R. J Am Chem Soc 2005; 127(10):3366-72), andpromote differentiation of cells into an osteogenic lineage (MatsumotoA, et al. (2006); LeGeros R Z. Clin Orthop Relat Res 2002(395):81-98;Bosnakovski D, Mizuno M, Kim G, Takagi S, Okumura M, Fujinaga T.Biotechnol Bioeng 2006; 93(6):1152-63; Mauney J R, Blumberg J, Pirun M,Volloch V, Vunjak-Novakovic G, Kaplan D L. Tissue Eng 2004;10(1-2):81-92; Phillips J E, Hutmacher D W, Guldberg R E, Garcia A J.Biomaterials 2006; 27(32):5535-45; Lu H H, El-Amin S F, Scott K D,Laurencin C T. J Biomed Mater Res A 2003; 64(3):465-74). Likewise,increasing the hydrophilicity of the biomaterial surface (Lee S J, KhangG, Lee Y M, Lee H B. J Biomater Sci Polym Ed 2002; 13(2):197-212; Park GE, Pattison M A, Park K, Webster T J. Biomaterials 2005; 26(16):3075-82;Miot S, Woodfield T, Daniels A U, Suetterlin R, Peterschmitt I, HebererM, et al. Biomaterials 2005; 26(15):2479-89; Yoo H S, Lee E A, Yoon J J,Park T G. Biomaterials 2005; 26(14):1925-33) or incorporating sulfatedpolymers (Chen Y L, Lee H P, Chan H Y, Sung L Y, Chen H C, Hu Y C.Biomaterials 2007; 28(14):2294-305; van Susante J L C, Pieper J, Buma P,van Kuppevelt T H, van Beuningen H, van Der Kraan P M, et al.Biomaterials 2001; 22(17):2359-69) can increase chondrocytedifferentiation and cartilage tissue formation.

The most commonly used chemical modification method for silk isderivatization of the carboxylic acid residues through carbodiimidecoupling with primary amines (Sofia, S. et al (2001); Vepari C P, KaplanD L. Biotechnol Bioeng 2006; 93(6):1130-7). However, only ˜1 mol % ofthe total amino acid content of the silk fibroin protein is composed ofaspartic and glutamic acid residues, thereby limiting the extent offunctionalization (Zhou, C Z., et al (2001). Reactions targetingtyrosine residues in silk have the potential to triple the amount offunctional group incorporation over carbodiimide coupling methods, as ˜5mol % of the amino acids in silk are tyrosines (Heslot, H. (1998); Zhou,C Z., et al (2001)). In addition, the tyrosine residues are distributedthroughout the protein sequence, allowing a homogeneous distribution ofmodifications along the scaffold protein (Zhou, C Z., et al (2001)). Afew strategies to modify the tyrosines in silk have been reported in theliterature involving cyanuric chloride-activated coupling (Gotoh Y,Tsukada M, Minoura N. Bioconjug Chem 1993; 4(6):554-9; Gotoh Y, TsukadaM, Minoura N, Imai Y. Biomaterials 1997; 18(3):267-71), enzyme catalyzedreactions with tyrosinase (Sampaio S, Taddei P, Monti P, Buchert J,Freddi G. J Biotechnol 2005; 116(1):21-33; Freddi G, Anghileri A,Sampaio S, Buchert J, Monti P, Taddei P. J Biotechnol 2006;125(2):281-94), or sulfation of the tyrosine residues withchlorosulfonic acid (Gotoh K, Izumi H, Kanamoto T, Tamada Y, NakashimaH. Biosci Biotechnol Biochem 2000; 64(8):1664-70; Tamada Y. Biomaterials2004; 25(3):377-83). However, these methods are limited in the varietyof molecules that can be incorporated.

The methods and compositions described herein, involve modification ofsilk tyrosine residues by a diazonium coupling reaction, wherein thetyrosine residues are functionalized by the addition of a side group.Functionalization of silk polymer can affect physical properties of asilk polymer including, but not limited to, hydrophobicity,hydrophilicity or time required for spontaneous gelation to occur. Someexamples of side groups used herein include, but are not limited to,sulfonic acid, carboxylic acid, amine, ketone or heptyloxy side groups.The extent of modification using several aniline derivatives can becharacterized using UV/vis and ¹H-NMR spectroscopy, and analysis of theresulting protein structure with ATR-FTIR spectroscopy. Introduction ofhydrophobic functional groups can facilitate rapid conversion of theprotein from a random coil to a β-sheet structure, while introduction ofhydrophilic groups can inhibit this process. An example method fordetermining the extent of silk modification is described in the Examplessection herein.

To summarize, multifunctional materials that can 1) directly interactwith hMSCs in a specific manner in order to induce differentiationtowards a particular cell lineage, or 2) provide an environment that isconducive to cell differentiation and tissue formation are contemplatedherein. As discussed above, silk has demonstrated utility as a cellscaffold for tissue engineering. The methods described herein allow thesurface chemistry of silk to be tailored in order to enhance formationof specific tissue types.

Formation of Modified Silk Polymer into an Insoluble State

When dissolved in aqueous solutions, silk is known to spontaneouslyassemble into a β-sheet structure at a relatively slow rate. Thephysical crosslinks formed by intermolecular β-sheet crystallizationresult in hydrogel formation. Depending on the protein concentration,this assembly can take weeks to occur but is catalyzed by lowering thepH or increasing the salt concentration (Kim, U J., et al (2004);Matsumoto, A. et al (2006). The transition from a random coil to aβ-sheet structure can also be induced by the addition of organicsolvents such as methanol to solid silk films or scaffolds.

Immersion in an alcohol or other suitable agent can produce aninsoluble, amorphous composition comprising primarily silk polymer as aβ-sheet structure (Sofia et al. Journal of Biomedical materials research2001, 54, 139-148). Therefore, silk structures are soaked in a β-sheetstructure inducing agent, such as alcohol, to induce the phasetransition to β-sheet structure. The type of a β-sheet structureinducing agent can be selected to generate structures with differentproperties. For example, when methanol and propanol are used to induceβ-sheet formation, the resulting structures are stronger but morebrittle and therefore suitable in bone regeneration. Various methods ofsilk insolubilization including insolubilization that does not rely uponorganic solvents or alcohol are contemplated herein and can be adaptedbased on the characteristics required for the modified silk describedherein. U.S. 20070187862 describes an all-aqueous approach to thepreparation of insoluble silk structures from solubilized silk fibroin.

Minerals

The methods described herein are suitable for tissue regeneration orstructural support of, for example bone and teeth, among others. Amineral component can be added to a scaffold, a structure or a surfacemade from modified silks described herein, for example in order toimprove biocompatibility or to enhance tissue regeneration whileproviding a support for injured or diseased tissue.

Minerals for use in the methods described herein, can include anybiocompatible mineral that one desires to use. In one particularembodiment, minerals are chosen that are similar to the minerals foundin bone or are important for bone growth/regeneration. A preferredmineral is hydroxyapatite, which has well-known characteristics withrespect to its compatibility with bone. Indeed, almost 70% of bone iscomprised of hydroxyapatite. Hydroxyapatite particles of varying sizecan be used to synthesize an implantable structure using the methodsdisclosed herein, and are available in nanocrystal, powder, granules andblocks from commercial sources such as Berkeley Advanced Biomaterials.Other minerals including silica, calcium, phosphate, or potassium, amongothers are contemplated for use herein.

Minerals can be added to the silk protein at any point during thediazonium chemistry process, provided that the mineral does notinterfere with modification of the silk protein, or the diazoniumchemistry does not alter the intended function of the mineral. Thus, theminerals can be added to the silk prior to attaching a chemical moietyor after attaching a chemical moiety. In addition, the minerals can bemixed into the silk protein prior to forming the silk into an insolublestate or can be sprayed onto the surface of an insoluble silk. Thoseskilled in the art can determine the method best suited for theapplication of the methods disclosed herein for the desired purpose ofthe modified silk.

Determining Hydrophilicity or Hydrophobicity

In some instances it is desired to tailor the physical properties of asilk protein for an intended purpose, for example culturing cells. Forexample, changes in hydrophobicity and hydrophilicity of a silk proteinare contemplated herein as a result of silk protein modification. Themodified silk can be tested for the desired hydrophobicity by thefollowing method.

Hydrophilicity (or hydrophobicity) can be measured by determining thecontact angle of a liquid on a hydrophilic (or hydrophobic surface). Thewater contact angle is determined by measuring the angle between asloping edge of a water droplet and the surface it rests on. Typically,very hydrophobic surfaces will increase the contact angle of water,since water is very hydrophilic and the surface tension of water isincreased. Conversely, very hydrophilic surfaces will decrease thecontact angle of water, allowing it to spread out along the surface.Thus, the higher the contact angle of water, the more hydrophobic asurface is. The modified silks disclosed herein can be measured usingthe silk as a surface with unknown hydrophobic or hydrophilicproperties. In this manner, the hydrophobic properties of the silk canbe estimated by changes in the contact angle of a water droplet on themodified silk surface.

In order to measure the hydrophilicity of a modified silk polymer, afilm is produced by casting a modified silk polymer onto a flat surface,followed by insolubilization of the silk, such that a surface ofmodified silk polymer is formed. A water droplet is placed onto the silkfilm after the formation of a modified silk into an insoluble state on aflat surface. The water contact angle is measured and then compared tothat for an unmodified silk polymer film produced in a similar manner oris estimated based on the known physical properties of water on avariety of surfaces with variable hydrophobicity. One embodiment of thewater contact method is described in Example 3, herein.

Surfaces for Modified Silk Polymer

In some cases it is useful to alter the properties of a surface bycoating it with a modified silk protein, such that the surface hasdesired physical properties. A surface can be any structure or portionof a structure that can be coated with a modified silk polymer, prior tothe formation of silk into an insoluble state. A surface can be composedof, for example, plastic, glass, ceramic or metal. It is preferred thata surface can withstand sterilization techniques such as autoclaving orUV radiation, especially in embodiments where the surface comprises animplantable structure or a tissue culture platform.

In some cases, the surface comprises an implantable structure such as amedical implant, for example an engineered support for bone. Thus, thesurface of the implant to be coated should be compatible with the tissuedomain into which it is to be implanted. Some non-limiting examples ofimplants that can be employed with the methods described herein includedrug delivery devices, stents, prostheses, dental implants, heartvalves, skin grafts, cosmetic implants, pacemakers, and infusion pumps.

In a preferred embodiment, the surface comprises a tissue cultureplatform including but not limited to wells, dishes, plates, discs,coverslips and slides. In this embodiment, the tissue culture surface iscoated with a modified silk polymer and then the silk polymer is formedinto an insoluble state, wherein the silk polymer coats a desiredportion of the surface. In another embodiment, a modified silk polymermay be coated onto or into an engineered scaffold of desired shape, forexample for ex vivo growth of organs. In this example cells can beseeded onto or into the scaffold and once the appropriate cell densityis achieved, the cells can be induced to differentiate. This approachhas wide-ranging capabilities for example, in the design or replacementorgans and can be varied for the specific needs of one skilled in theart. Tailor made silk platforms can provide a surface amenable to theproperties required to efficiently culture a wide range of cell types.

In addition, the modified silk can be formed into an insoluble state,such that the silk itself is the surface, for example for cell culture.Thus the surface can be comprised of a silk that is formed into aninsoluble state in a mould, which is then removed such that the modifiedsilk retains the 3-dimensional structure of the mould. Modified silksused in this manner are also contemplated herein.

Implantable Structures

An “implantable structure” is generally any structure that uponimplantation does not generate an immune response in a host organism.Thus, an implantable structure should not for example, be or contain anirritant, or contain LPS etc. In addition, in some instances, it ispreferred that an implantable structure does not prohibit cellinfiltration, blood vessel growth or other properties that would inhibitbioresorption or integration of the structure into tissue. For suchinstances, for example, it is important that the structure is not simplya solid 3-dimensional form but comprises or develops some porosity suchthat cells, etc., can gain access during the resorption process (thatis, unless the lack of bioresorption is desired). While it is generallypreferred that an implantable structure does not raise or provoke animmune response (e.g., inflammation or the raising of antibodies againsta component of an implant), in some cases it can be beneficial for thestructure to induce an immune-response (e.g., generation of antibodiesagainst a specific antigen) or to prevent integration into tissue. Suchaspects are also contemplated within the methods described herein.

An implantable structure can be applied to a wide variety of uses,however it is preferred that an implantable structure, as describedherein, is implanted for the repair or support of bone or toothstructures. Implantable silk structures can be used e.g., to givetemporary support for teeth, for broken bones, for fragile/weak bones,or to speed healing of bone fractures, breaks, loss of calcification,etc. Modified silk coated structures as described herein can also beused to deliver a bioactive agent, either as a primary use or secondaryto repair or support of a tissue. Bioactive agents useful in suchembodiments are described herein below.

In general, the length of a tissue regenerative growth period willdepend on the particular tissue being implanted with a silk structure.The growth period can be continued until the new tissue has attaineddesired properties, e.g., until the regenerating tissue has reached aparticular thickness, size, strength, composition of proteinaceouscomponents, and/or a particular cell density. Methods for assessingthese parameters are known to those skilled in the art. The implantablestructure should reabsorb at a rate that does not exceed the growthperiod of the tissue. Thus, the structure should remain substantiallyintact until sufficient infiltration of surrounding tissue occurs (asdetected by methods known in the art) and the implantable structure isno longer necessary for tissue strength or structure (e.g., bonedensity). Such agents can provide prophylactic or therapeutic benefiteither in situ, e.g., through promotion of desired biological processes,or, e.g., by leaching out of the structure after implantation.

Bioactive Agents

In some cases it is preferable to incorporate a bioactive agent into amodified silk polymer. A bioactive agent can be incorporated into oronto a modified silk coating of a medical implant, for example toencourage local tissue growth, prevent infection, or promoteangiogenesis. A bioactive agent can be incorporated onto or into amodified silk coating of a tissue culture platform for example, toenhance cell growth, to speed cell division, to prevent bacterialinfiltration of mammalian cells, to maintain an undifferentiated stateor to induce cells to differentiate into a desired lineage.

In one preferred embodiment, additives such aspharmaceutical/therapeutic agents, or biologically active agents, areincorporated into a modified silk polymer. For example, growth factors,pharmaceuticals, or biological components can be incorporated into thepolymer prior to, or following formation of silk into an insolublestate. Any pharmaceutical carrier can be used that does not dissolve orotherwise interfere with the solidified modified silk. The bioactive ortherapeutic agents can be added to the modified silk as a liquid, afinely divided solid, or any other appropriate physical form.Alternatively, a bioactive agent can be added to a pre-formed insolublemodified silk polymer as described herein by immersing the insolublesilk polymer in a solution comprising a bioactive agent. That is, theagent need not necessarily be present when the modified silk is formedinto an insoluble state. The bioactive agent can also be chemicallybonded to the silk using a chemical coupling procedure appropriate forthe bioactive agent used.

The variety of different pharmaceutical/therapeutic agents that can beused in conjunction with the methods described herein is wide andincludes, but is not limited to, small molecules, proteins, antibodies,peptides and nucleic acids. In general, bioactive agents which can beadministered via the invention include, without limitation:anti-infectives such as antibiotics and antiviral agents;chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents;analgesics and analgesic combinations; anti-inflammatory agents;hormones such as steroids; growth factors (bone morphogenic proteins(i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GFD-5, GFD-7 andGFD-8), epidermal growth factor (EGF), fibroblast growth factor (i.e.FGF 1-9), platelet derived growth factor (PDGF), insulin like growthfactor (IGF-I and IGF-II), transforming growth factors (i.e. TGF-β-III),vascular endothelial growth factor (VEGF)); anti-angiogenic proteinssuch as endostatin, and other naturally derived or geneticallyengineered proteins, polysaccharides, glycoproteins, or lipoproteins.Growth factors important for, e.g., bone growth, are described in “TheCellular and Molecular Basis of Bone Formation and Repair” by VickiRosen and R. Scott Thies, published by R. G. Landes Company,incorporated herein by reference. Additionally, the modified silkdescribed herein can be used to deliver any type of molecular compound,such as, pharmacological materials, vitamins, sedatives, steroids,hypnotics, antibiotics, chemotherapeutic agents, prostaglandins, andradiopharmaceuticals. The silk coated surfaces described herein aresuitable for delivery of the above materials and others including butnot limited to proteins, peptides, nucleotides, carbohydrates, simplesugars, cells, genes, anti-thrombotics, anti-metabolics, growth factorinhibitor, growth promoters, anticoagulants, antimitotics,fibrinolytics, anti-inflammatory steroids, and monoclonal antibodies.

Examples of other biologically active agents suitable for use in themethods described herein include, but are not limited to: cellattachment mediators, such as collagen, elastin, fibronectin,vitronectin, laminin, proteoglycans, or peptides containing knownintegrin binding domains e.g. “RGD” integrin binding sequence, orvariations thereof, that are known to affect cellular attachment(Schaffner P & Dard 2003 Cell Mol Life Sci. January; 60(1):119-32;Hersel U. et al. 2003 Biomaterials November; 24(24):4385-415);biologically active ligands; and substances that enhance or excludeparticular varieties of cellular or tissue ingrowth. Such additives areparticularly useful in tissue engineering applications where, forexample, structures are engineered in vitro to include cells that imparta beneficial characteristic on the structure to be implanted. Forexample, the steps of cellular population of a 3-dimensionalsilk-hydroxyapatite scaffold matrix preferably are conducted in thepresence of growth factors effective to promote proliferation of thecultured cells employed to populate the matrix. Agents that promoteproliferation will be dependent on the cell type employed. For example,when fibroblast cells are employed, a growth factor for use herein maybe fibroblast growth factor (FGF), most preferably basic fibroblastgrowth factor (bFGF) (Human Recombinant bFGF, UPSTATE Biotechnology,Inc.). Other examples of additive agents that enhance proliferation ordifferentiation include, but are not limited to, osteoinductivesubstances, such as bone morphogenic proteins (BMP); cytokines, growthfactors such as epidermal growth factor (EGF), platelet-derived growthfactor (PDGF), insulin-like growth factor (IGF-I and II) TGF-α and thelike.

Modified silk coated structures can be used to deliver therapeuticagents to cells and tissues. The ability to incorporate, for examplepharmaceutical agents, growth factors and other biological regulators,enzymes or possibly even cells in a modified silk coated structuredescribed herein provides for stabilization of these components for longterm release and stability, as well as better control of activity andrelease.

Other reagents, necessary or indicated for assisting the stability oractivity of a bioactive agent can also be included in the mixtures usedto create a modified silk with bioactive agents as described herein.Thus, buffers, salts or co-factors can be added as necessary.

Cells

Any type of cell can be utilized in the methods described herein. It ispreferable that the cells comprise stem cells, which can be induced todifferentiate into a desired lineage, however differentiated cells ofany desired lineage can also be used and are contemplated herein.

In one embodiment, the modified silk polymer is coated onto or into amedical implant. A number of different cell types or combinationsthereof may be employed in this embodiment, depending upon the intendedfunction of the medical implant being produced. These cell typesinclude, but are not limited to: smooth muscle cells, skeletal musclecells, cardiac muscle cells, epithelial cells, endothelial cells,urothelial cells, fibroblasts, myoblasts, chondrocytes, chondroblasts,osteoblasts, osteoclasts, keratinocytes, hepatocytes, bile duct cells,pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamic,pituitary, ovarian, testicular, salivary gland cells, adipocytes, andprecursor cells. For example, smooth muscle cells and endothelial cellsmay be employed for muscular, tubular implants, e.g., implants intendedas vascular, esophageal, intestinal, rectal, or ureteral implants;chondrocytes may be employed in cartilaginous implants; cardiac musclecells may be employed in heart implants; hepatocytes and bile duct cellsmay be employed in liver implants; epithelial, endothelial, fibroblast,and nerve cells may be employed in implants intended to function asreplacements or enhancements for any of the wide variety of tissue typesthat contain these cells. In general, any cells may be employed that arefound in the natural tissue to which the implant is intended tocorrespond. In addition, progenitor cells, such as myoblasts or stemcells, can be employed to produce their corresponding differentiatedcell types. In some instances it may be preferred to use neonatal cellsor tumor cells.

Cells can be obtained from donors (allogenic) or from recipients(autologous). Cells can also be of established cell culture lines, oreven cells that have undergone genetic engineering. Pieces of tissue canalso be used, which may provide a number of different cell types in thesame structure.

Appropriate in vitro growth conditions for mammalian cells are wellknown in the art (Freshney, R. I. (2000) Culture of Animal Cells, aManual of Basic Technique. Hoboken N.J., John Wiley & Sons; Lanza et al.Principles of Tissue Engineering, Academic Press; 2nd edition May 15,2000; and Lanza & Atala, Methods of Tissue Engineering Academic Press;1st edition October 2001). Cell culture media generally includeessential nutrients and, optionally, additional elements such as growthfactors, salts, minerals, vitamins, etc., that may be selected accordingto the cell type(s) being cultured. Particular ingredients may beselected to enhance cell growth, differentiation, secretion of specificproteins, etc. In general, standard growth media includes, for exampleDulbecco's Modified Eagle Medium, low glucose (DMEM), with 110 mg/Lpyruvate and glutamine, supplemented with 10-20% fetal bovine serum(FBS) or calf serum and 100 U/ml penicillin is appropriate as arevarious other standard media well known to those in the art. Growthconditions will vary dependent on the type of mammalian cells in use andtissue desired.

The cells that are used for methods of the present invention should bederived from a source that is compatible with the intended recipient.The cells are dissociated using standard techniques and seeded onto orinto the scaffold of the implant. In vitro culturing optionally may beperformed prior to implantation. Alternatively, the scaffold isimplanted into the subject, allowed to vascularize, then cells areinjected into the scaffold. Methods and reagents for culturing cells invitro and implantation of a tissue scaffold are known to those skilledin the art.

Uniform seeding of cells is preferable. In theory, the number of cellsseeded does not limit the final tissue produced, however optimal seedingmay increase the rate of generation. The number of seeded cells can beoptimized using dynamic seeding (Vunjak-Novakovic et al. BiotechnologyProgress 1998; Radisic et al. Biotechnoloy and Bioengineering 2003).

In one embodiment, silk matrix scaffolds are seeded with multipotentcells in the presence of media that induces either bone or cartilageformation. Suitable media for the production of cartilage and bone arewell known to those skilled in the art. As used herein, “multipotent”cells have the ability to differentiate into more than one cell type inresponse to distinct differentiation signals. Examples of multipotentcells include, but are not limited to, bone marrow stromal cells (BMSC)and adult or embryonic stem cells. In a preferred embodiment BMSCs areused. BMSCs are multipotential cells of the bone marrow which canproliferate in an undifferentiated state and with the appropriateextrinsic signals, differentiate into cells of mesenchymal lineage, suchas cartilage, bone, or fat (Friedenstein, A. J. 1976. Int Rev Cytol47:327-359; Friedenstein et al. 1987. Cell Tissue Kinet 20:263-272;Caplan, A. I. 1994. Clin Plast Surg 21:429-435; Mackay et al. 1998.Tissue Eng 4:415-428; Herzog et al. Blood. 2003 Nov. 15;102(10):3483-93. Epub 2003 Jul. 31).

In addition, cells grown on a scaffold for the purpose of growingorgans/tissues in an in ex vivo manner are also contemplated herein.

Differentiation of Stem Cells

Stem cells can be differentiated or maintained in an undifferentiatedstate by a number of methods known to those skilled in the art.Differentiation can be induced for example, by the addition of achemical or biological agent (e.g, cytokines), genetic manipulation ofcell-type specific markers, or addition of specific growthfactors/substrates to culture media. Genetic manipulation can includefor example, the over-expression of cell-type specific markers to inducestem cell differentiation into a desired lineage, or the inhibition ofstem cell specific markers by RNA interference or targeted geneablation.

In one embodiment, methods are provided for producing bone or cartilagetissue in vitro comprising culturing multipotent cells on a porousmodified silk scaffold under conditions appropriate for inducing bone orcartilage formation. Suitable conditions for the generation of bone andcartilage are well known to those skilled in the art. For example,conditions for the growth of cartilage tissue often comprisenonessential amino acids, ascorbic acid-2-phosphate, dexamethasone,insulin, and TGF-β1. Suitable conditions for the growth of bone ofteninclude ascorbic acid-2-phosphate, dexamethasone, β-glycerolphosphateand BMP-2. In a preferred embodiment, ascorbic acid-2-phosphate ispresent at a concentration of 50 ug/ml, dexamethasone is present at aconcentration of 10 nM, α-glycerolphosphate is present at aconcentration of 7 mM and BMP-2 is present at a concentration of 1ug/ml. The formation of cartilaginous tissue or bone can be monitored byassays well known to those in the art including, but not limited to,histology, immunohistochemistry, and confocal or scanning electronmicroscopy (Holy et al., J. Biomed. Mater. Res (2003) 65A:447-453).

Optics

In addition to the use of modified silks in implantable devices andtissue culture platforms, the modified silks disclosed herein can alsobe used in optical applications such as those described inPCT/US07/83642, PCT/US07/83600, PCT/US07/83620, PCT/US07/83634,PCT/US07/83639, and PCT/US07/83646, which are incorporated herein intheir entirety. The ability of the modified silks, as described herein,to be tailored for specific biochemical and physical properties allowsfor a high degree of flexibility in optical applications such asdiffraction gratings, pattern generators and lenses. Various opticalapplications for use with the modified silks described herein arecontemplated.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose skilled in the art, can be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

EXAMPLES Example 1 An Exemplary Method for Modifying a Silk Polymer Witha Diazonium Salt Materials and Methods

All chemicals were purchased from Aldrich, Sigma or Fluka and usedwithout further purification. Cell medium ingredients were purchasedfrom Invitrogen and Sigma. Cocoons from B. mori silkworm were obtainedfrom Tajima Shoji Co, (Yokohama, Japan). UV-Vis data were measured witha GBC 916 spectrophotometer in water unless otherwise indicated.Infrared spectra were measured on solid films in ambient atmosphere withan Equinox 55 ATR-FTIR (Bruker, Billerica, Mass.) using an AttenuatedTotal Reflectance (ATR) accessory. 1H-NMR spectra were recorded with aBruker Avance 400 NMR Spectrometer using D2O as the solvent.

Preparation of Aqueous Silk Solutions

Aqueous solutions of the silk protein were obtained using previouslypublished methods from Altman et al, (2003); H-J. Jin J. P., et al(2005); Nazarov R., et al (2004), which are incorporated herein byreference. Cocoons from the B. mori silkworm were cut and boiled for 1hour in an aqueous solution of 0.02 M Na₂CO₃, rinsed once with boilingwater, then three times with distilled water. The purified silk fibroinwas then solubilized by dissolving in a 9 M LiBr solution at 60° C. for45 minutes giving a 20 wt % solution. This solution was filtered througha 5 gm syringe filter, then dialyzed against distilled water for 2 days,changing the water 3 times, then against borate buffer (100 mM borate,150 mM NaCl, pH 9) for an additional day, changing the buffer 2 times.These silk solutions had a final concentration of ˜9-11 wt % and couldbe stored in a refrigerator for at least 6 months.

Diazonium Coupling Reaction with Silk

As illustrated in FIG. 1, diazonium reactions with silk involve anelectrophilic aromatic substitution reaction between the tyrosinephenolic side chains and a diazonium salt resulting in an azobenzenederivative (Kim, M S., et al (2007); Hutmacher, D W. (2000); Pielak G.J. et al., Biochemistry, 23:589-596 (1984), Tabachnick M. et al., J.Biol. Chem., 234(7):1726-1730 (1959). Histidine residues can also bemodified with this chemistry, but the contribution here is negligible ashistidine comprises <1% of the amino acid content of silk (Zhou, C Z.,et al (2001)).

While there are many more commercially available derivatives, theanilines shown in FIG. 2 were used here to demonstrate the range offunctional groups that can be incorporated into silk using thischemistry. These anilines contain carboxylic acid (1), amine (2), ketone(3), sulfonic acid (4), and alkyl (5) functional groups.

A. General Procedure:

A cooled solution of 1.25 mL of a 0.2 M acetonitrile solution of anilineand 625 μL of a 1.6 M aqueous solution of p-toluenesulfonic acid, werecombined with a cooled aqueous solution of 0.8 M NaNO₂. The mixture wasvortexed briefly, and then placed in an ice bath for 15 minutes. In atypical experiment, the total reaction volume was 1 mL, where 850 μL ofthe silk solution was combined with 0-150 μL of the stock diazonium saltsolution. In cases where less than 150 μL of the diazonium salt stockwas used, the solution was diluted with a mixture of 1:1acetonitrile/water to give a total volume of 150 μL. After combining thesilk and diazonium salt, the reaction was allowed to proceed for 5-30minutes, then purified by passing the reaction mixture throughdisposable Sephadex size exclusion columns (NAP-10, GE Healthcare),pre-equilibrated with distilled water. To prepare samples for ¹H-NMR,the reaction mixture was eluted through Sephadex columnspre-equilibrated with deuterium oxide (D₂O). In all cases >90% of themodified protein was recovered after the reaction.

B. Silk Fibroin Control:

For UV and NMR comparisons, the silk solution in borate buffer wasdesalted by passing the solution through a Sephadex size exclusioncolumn pre-equilibrated with distilled water or D₂O. UV/Vis:λ_(max (pH 7))=215 nm (carbonyl) and 275 nm(tyrosine)/λ_(max (pH 14))=240 nm (carbonyl) and 290 nm (tyrosine). ¹HNMR (400 MHz): δ 0.85 (br, 3H, Valγ), 1.16-1.18 (br, 1H), 1.35 (m, 12H,Alaβ), 2.02 (br, 1H, Valβ), 2.88-2.99 (br, 2H, Asp/Tyrβ), 3.80-3.93 (m,16H, Serβ/Glyα), 4.26-4.30 (m, 5H, Alaα), 4.41-4.48 (m, 3H, Serα),6.73-6.77 (m, 2H, Tyrφ), 6.95-7.03 (m, 2H Tyrφ)), 7.14-7.28 (br, 1H,Trpφ)).

C. 4-Aminobenzoic acid Derivative (Azosilk-1):

The diazonium salt was allowed to react with the silk for 20 minutesprior to purification. UV/vis: λ_(max (pH 7))=329 nm(azo)/λ_(max (pH 14))=327 and 485 nm (azo). ¹H NMR (400 MHz): 0.85 (br,3H, Valγ), 1.16-1.18 (br, 1H), 1.35 (s, 12H, Alaβ), 2.02 (br, 1H, Valβ),2.88-2.99 (br, 3H, Asp/Tyrβ), 3.80-3.93 (m, 17H, Serβ/Glyα), 4.26-4.30(m, 5H, Alaα), 4.41-4.48 (m, 3H, Serα), 6.71 (br, 1.5H, Tyrφ), 6.98 (br,1.5H, Tyrφ), 7.10-7.69 (br, 2H, azo), 7.83 (br, 1H, azo).

D. 4-(2-Aminoethyl)Aniline Derivative (Azosilk-2):

The diazonium salt was allowed to react with the silk for 10 minutesprior to purification. Longer reaction times resulted in proteingelation. UV/vis: λ_(max (pH 7))=328 nm (azo). Due to low conversion tothe azo derivative, no new peaks were observed in the ¹H NMR spectra.

E. 4′-Aminoacetophenone Derivative (Azosilk-3):

The diazonium salt was allowed to react with the silk for 5 minutesprior to purification. Longer reaction times resulted in proteingelation. UV/vis: λ_(max (pH 7))=333 nm (azo)/λ_(max (pH 14))=336 and505 nm (azo). Rapid protein gelation prevented acquisition of ¹H NMRspectra.

F. 4-Sulfanilic Acid Derivative (Azosilk-4):

4-sulfanilic acid was dissolved in water instead of acetonitrile. Thediazonium salt was allowed to react with the silk for 20 minutes priorto purification. UV/vis: λ_(max (pH 7))=325 nm (azo)/λ_(max (pH 14))=327and 486 nm (azo). ¹H NMR (400 MHz): 0.85 (br, 3H, Valγ), 1.16-1.18 (br,1H), 1.35 (s, 12H, Alaβ), 1.95-2.11 (br, 1H, Valβ), 2.88-2.99 (br, 2H,Asp/Tyrβ), 3.80-3.93 (m, 20H, Serβ/Glyα), 4.26-4.30 (m, 6H, Alaα),4.41-4.48 (s, 3H, Serα), 6.71 (br, 2H, Tyrφ), 6.98 (br, 2H, Tyrφ),7.35-7.67 (br, 1H, azo), 7.8 (br, 0.75H, azo).

G. 4-(Heptyloxy)Aniline Derivative (Azosilk-5):

The diazonium salt was allowed to react with the silk for 5 minutesprior to purification. Longer reaction times result in protein gelation.UV/vis: λ_(max)=315 nm (azo)/λ_(max (pH 14))=323 and 481 nm (azo). Dueto low conversion to the azo derivative, no new peaks were observed inthe ¹H NMR spectra.

Statistical Analysis

Statistical differences between samples were evaluated in Excel using atwo-sided Student's t-test. Values with p<0.05 were consideredsignificant.

Example 2 An Exemplary Method for Culturing Cells on Diazonium ModifiedInsoluble Silk Polymer Preparation of Silk Films for Cell Culture

All five modified silk fibroin solutions were prepared as describedabove by combining 375 μL of each diazonium salt with 2 mL of silksolution in borate buffer diluted with 125 μL of a 1:1acetonitrile/water mixture (approximately 0.40 equivalents of diazoniumsalt relative to the number of tyrosines in silk). The unmodified silkcontrol was prepared by diluting 2 mL of silk solution in borate bufferwith 500 μL of a 1:1 acetonitrile/water mixture. The modified andunmodified silk solutions were purified by passing the reaction mixturethrough disposable Sephadex size exclusion columns, pre-equilibratedwith ultrapure water. To ensure that all small molecules and salts wereremoved, these solutions were passed through a second Sephadex sizeexclusion column, again eluting with ultrapure water. The resultingsolutions had a silk concentration of ˜2 wt % in water.

The silk solutions were sterilized using a 0.22 μm syringe filter in atissue culture hood. Twenty-four-well tissue culture plates were coatedwith 300 μL of the modified silk solutions, as well as unmodified silk,and left to dry overnight in a laminar flow hood. To make the silkinsoluble in water, the films were then soaked in 1 mL of a 70% methanolsolution in water for 2 hours and then dried overnight. Prior to cellseeding, 1 mL of cell culture medium was added to each well, soaked for30 minutes, and then aspirated.

Cell Culture

Bone marrow aspirate from a 20-year old male was obtained from CambrexBioscience (Walkersville, Md.). hMSCs were isolated from the aspirate bytheir ability to adhere to tissue culture plates. The marrow was dilutedwith growth medium containing high glucose Dulbecco's modified eaglemedium (DMEM) supplemented with 10% fetal bovine serum, 1%antibiotic/antimycotic, 1% non-essential amino acids, and 10 ng basicfibroblast growth factor (bFGF) and was plated in tissue culture plates.The plates were kept in a humidified incubator at 37° C. and 5% CO2. At90% confluency, the marrow was removed and the cells were detached andreplated at a density of 5,000 cells/cm² giving passage 1 cells (P1).When 90% confluent, cells were detached and frozen in liquid nitrogenuntil ready for use (P2). In subsequent experiments, hMSCs were culturedeither in growth medium described above, or osteogenic medium containinghigh glucose Dulbecco's modified eagle medium (DMEM) supplemented with10% fetal bovine serum, 1% antibiotic/antimycotic, 1% non-essentialamino acids, 100 nM dexamethasone, 10 mM α-glycerolphosphate, and 0.05mM L-ascorbic acid 2-phosphate. The medium was changed every 3-4 days.

P2 cells described above were thawed, suspended in medium, and platedonto the silk films at a density of 5,000 cells/well in a 24-well plate.Cells were incubated in growth medium until they reached 80% confluence,at which time half of the wells were switched to osteogenic medium whilethe other half was maintained in growth medium for one week. The cellgrowth and shape were monitored using a phase contrast light microscope(Carl Zeiss, Jena, Germany) equipped with a Sony Exwave HAD 3CCD colorvideo camera.

Example 3 Characterization of Exemplary Modified Silk Polymers

Carboxylic acid (1), amine (2), ketone (3), sulfonic acid (4), and alkyl(5) functional groups were used to demonstrate the range of functionalgroups that can be incorporated into silk using this chemistry. Puresilk fibers were dissolved in concentrated lithium bromide, and dialyzedinto borate buffer prior to reaction. Silk fibroin contains about 280tyrosine amino acids per protein (Zhou C Z, et al. Proteins 2001;44(2):119-22), so the molar ratio of diazonium salt to tyrosine wastailored to produce the desired level of modification. Reaction timesvaried for the different anilines used, but in all cases >90% of themodified protein was recovered after the reaction.

Characterization of Azo Incorporation

The silk protein was treated with 0.10, 0.25 or 0.40 molar equivalentsof the diazonium salt of (4) relative to the number of tyrosineresidues. These samples were analyzed with UV/vis and ¹H-NMRspectroscopy and compared with unmodified silk (FIG. 3). As theequivalents increase, the tyrosine absorption at 280 nm in the nativeprotein decreases (FIG. 3 a). Likewise, a strong absorptioncorresponding to the newly formed azobenzene chromophore can be seen at325 nm with a shoulder at 390 nm (Pielak G J, Mickey U S, Kozo I, Legg JI. Biochemistry 1984; 23:589-596. These absorptions can be assigned tothe azobenzene π-π* and n-π* transitions, respectively (FIG. 3 a).

¹H NMR spectra of silks treated with the diazonium salt of (4) alsodemonstrate increasing incorporation of the azo moiety, as shown in FIG.3 b. With increasing diazonium salt equivalents, the original tyrosinepeaks shift up-field and broaden while peaks consistent with the newaromatic ring in the azo group grow in (Tamada, Y. et al (2004); AntonyM J, Jayakannan M. J Phys Chem B 2007; 111(44):12772-80; Bundi A,Wuthrich K. Biopolymers 1979; 18:285-297).

For each condition, the number of azo-modified tyrosines in each silkmolecule was estimated using Beer's Law (FIG. 3 c). The concentration ofazo groups was calculated from the absorbance at 325 nm using anextinction coefficient of 22,000 M⁻cm⁻¹ (Pielak G J, Mickey U S, Kozo I,Legg J I. Biochemistry 1984; 23:589-596; Tabachnick M, Sobotka H. J BiolChem 1959; 234(7):1726-30). Silk contains approximately 280 tyrosinesper molecule (Zhou, C Z. et al (2001)), so the percentage of modifiedtyrosines in a silk solution of known concentration can be calculated.As outlined in FIG. 3 c, the number of tyrosines converted to azoderivatives was found to scale linearly with increasing diazonium saltequivalents of (4), where approximately 70% of the added diazonium saltresults in azo formation.

The spectra for silks modified with (4) are shown in FIG. 3 andillustrate that the extent of protein modification was highly controlledwith all of the aniline derivatives used. However, differences inreactivity were observed, particularly when using derivative (2) and(5). The diazonium coupling reaction favors anilines containingelectron-withdrawing groups, such as carboxylic and sulfonic acids.Aniline derivatives (2) and (5) contain electron-donating substituentsthat lower the reactivity considerably.

FIG. 4 a compares the UV/vis spectra of unmodified and modified silks inwater at pH 7. Spectra were normalized to the carbonyl peak at 210 nm,as the contribution to the absorbance from the carbonyls in derivatives(1), (3) and (4) was found to be negligible when compared to thecontribution from the amide bonds in the protein backbone. The silkswere each treated with 0.4 equivalents of diazonium salt relative to thenumber of tyrosine residues. Addition of more equivalents of (2), (3),and (5) results in immediate gelation of the protein, so 0.4 eq was usedto compare reactivity of all the derivatives side-by-side. Thecarboxylic acid azosilk-1, ketone azosilk-3, and sulfonic acid azosilk-4exhibit strong absorptions for the azobenzene π-π* transition at 331,325, and 332 nm, respectively (Pielak, G J. et al (1984); Tabachnick, M.et al (1959); Dabbagh H A, Teimouri A, Chemahini A N. Dyes and Pigments2006; 73(2):239-244; Nakayama K, Endo M, Majima T. Bioconjug Chem 2005;16(6):1360-6). Little or no absorption was seen at 275 nm for the nativetyrosine residues. In contrast, the amino azosilk-2 and heptyloxyazosilk-5 displayed smaller peaks at 329 and 315 nm for the azo moiety,respectively, and still had significant tyrosine absorption at 275 nm.This indicates that the conversion to the azo derivative was much lowerfor these anilines containing electron-donating substituents.

The number of azo-modified tyrosines in each silk molecule was estimatedusing Beer's Law (FIG. 4 b). Reported extinction coefficients range from20-22,000 M⁻¹cm⁻¹ for similar azobenzenes (Tabachnick, M. et al (1959);Dabbagh H A, Teimouri A, Chemahini A N. Dyes and Pigments 2006;73(2):239-244; Nakayama K, et al (2005)), so for simplicity 22,000M⁻¹cm⁻¹ was used here for all derivatives. Using these values it wasestimated that reaction with 0.4 equivalents of the electron-deficientanilines (1), (3) and (4) results in ˜30% conversion of the tyrosines toazo groups. In contrast, the electron-rich anilines (2) and (5) resultin ˜10% conversion.

FIG. 4 shows that the differences in reactivity were further confirmedwith ¹H NMR. Peaks corresponding to the azo derivative were onlyobserved in the spectra of carboxylic acid azosilk-1 and sulfonic acidazosilk-4 which had the highest levels of conversion. Low incorporationof the azo groups in amino azosilk-2 and heptyloxy azosilk-5 made thespectra indistinguishable from unmodified silk. From the UV/vis data,the ketone azosilk-3 also had high levels of conversion to the azoderivative. However, the samples gelled rapidly at the concentrationsneeded for NMR, preventing clear resolution of the peaks.

Contact Angle Measurements

The change in the overall hydrophilicity of the silk following reactionwith each of these aniline derivatives was quantified using watercontact angle measurements. The silk solutions were treated with 0.4equivalents of diazonium salt, and cast onto a glass slide. After dryingovernight, the silk films were treated with methanol to make the filmsinsoluble in water.

As outlined in Table 1, films of unmodified silk fibroin have a watercontact angle of ˜60°. Amino azosilk-2 was found to have a similarcontact angle to native silk, which is likely due to the lowincorporation of the azo moiety (˜30%). The carboxylic acid azosilk-1and sulfonic acid azosilk-4 both had high levels of azo incorporation,but only the sulfonic acid derivative dramatically lowered the contactangle to 43±5°. Conversely, reaction of silk with the ketone (3) andheptyloxy (5) aniline derivatives resulted in an increase in contactangle due to incorporation of hydrophobic residues. It is interesting tonote that even though the extent of reaction for derivative (5) was low(as discussed in the previous section) it still resulted in a dramaticincrease in the hydrophobicity of the silk.

Infrared Spectroscopy (FTIR) Analysis of Hydrogel β-Sheet Structure

Modification of silk with the hydrophobic ketone (3) and heptyloxy (5)derivatives was found to promote β-sheet formation of the silk protein,resulting in rapid hydrogel formation. Reaction with aniline (2) alsodecreased the gelation time to 1-2 days. Conversely, carboxylic acidazosilk-1 and especially sulfonic acid azosilk-4 exhibited a markeddecrease in the propensity for β-sheet formation. Samples of sulfonicacid azosilk-4 in solution have been stored at room temperature for >1year, and show no signs of protein aggregation or gelation. Forcomparison, unmodified silk samples from the same batch were found togel at room temperature within 1 month. While spontaneous β-sheetformation of sulfonic acid azosilk-4 in solution is inhibited, the silkis still able to form a β-sheet structure when dehydrated with organicsolvents such as methanol.

The propensity of the azosilk derivatives to form a β-sheet structurecan be characterized using FTIR spectroscopy (Matsumoto, A., et al(2006); Venyaminov S, Kalnin N N. Biopolymers 1990; 30(13-14):1259-71;Hu X, Kaplan D L, Cebe P. Macromolecules 2006; 39:6161-6167). Thetransition from a random coil to a β-sheet structure can be detected bymonitoring the N—H stretch, the N—H bend and the C—N stretch in the FTIRspectrum. The peak corresponding to the N—H stretch bond vibration canbe found at 1650 cm⁻¹ for proteins that exhibit a random coil structure.When proteins assume a β-sheet structure, the amide hydrogensparticipate in hydrogen bonding which shifts this peak to ˜1625 cm⁻¹.Similarly, the N—H bend peak will shift from 1540 to 1520 cm⁻¹, and theC—N stretch will shift from 1230 to 1270 cm⁻¹ upon β-sheet formation.

The FTIR spectra for cast films of each of the modified silks are shownin FIG. 4. After treatment with 0.4 equivalents of diazonium saltrelative to the number of tyrosine residues, purified solutions of theazosilks in water (3 wt %) were cast into films and dried. Ketoneazosilk-3 and heptyloxy azosilk-5 at this concentration and modificationlevel spontaneously form hydrogels within 30 minutes, so films of thesesilk derivatives exhibit shifts in the FTIR spectra consistent withβ-sheet formation. Carboxylic acid azosilk-1, amino azosilk-2 andsulfonic acid azosilk-4 all have spectra similar to unmodified silk in arandom coil conformation.

FIG. 5 shows that while the FTIR spectra were useful for characterizingthe structural conformation of the silk proteins, observation of thepeaks corresponding to the new functional groups installed through thediazonium reaction could not be observed, as the spectra were dominatedby peaks corresponding to the protein backbone.

hMSC Proliferation and Differentiation on Azosilk Films

After demonstrating that the modification of silk through this diazoniumcoupling strategy can change the material hydrophilicity and structure,the ability of these azosilk derivatives to support hMSC growth anddifferentiation was investigated. For these studies, tissue cultureplates were coated with solutions of the various azosilk derivatives,dried, and treated with methanol to render the films insoluble in water.hMSCs were grown on these substrates and differentiated into anosteogenic lineage. Proliferation, morphology and gene expression wereanalyzed and compared to cells grown on tissue culture plastic (TCP) orunmodified silk.

Cell Proliferation

On day 4, 7, 9 and 12 cell metabolic activity was quantified using thealamarBlue® assay (Invitrogen, #DAL1100) according to the manufacturer'sinstructions. Briefly, 1 mL of a solution containing basic medium (DMEMsupplemented with 1% antibiotic/antimycotic and 10% FBS) with 10%alamarBlue® solution was added to 3 wells from each type of silk film orTCP, and incubated for 2 hours. A 100 μA aliquot was then taken fromeach well, and analyzed for fluorescence exciting at 560 nm andrecording the emission at 590 nm. Background fluorescence from thealamarBlue® solution alone was subtracted, and the sample values from 3wells of each culture were averaged.

The relative proliferation rates of hMSCs grown on the differentsubstrates were monitored using the alamarBlue® assay. As shown in FIG.6, hMSC growth rates on the various silk derivatives were consistent upto day 7, but significant differences became apparent by day 9. Ingeneral, cells proliferated extensively up to day 9 and then exhibitedonly small increases or decreases in metabolic activity from day 9 today 12. However, cells grown on the hydrophobic heptyl azosilk-5 had aslower, but increasing growth rate up to day 12. Comparison of the datafrom the final time point showed that there were significantly morecells on the carboxylic acid azosilk-1 and sulfonic acid azosilk-4 thanon the remaining silk derivatives.

Cell Morphology

Cell viability in the monolayer cultures was assessed using a Live/Deadassay kit (Invitrogen # L-3224). The staining solution was prepared byadding 10 uL of the ethidium homodimer-1 solution to 5 mL PBS, followedby 5 uL calcein AM. The medium was aspirated from the cell wells, andwashed gently with PBS 2×. Two hundred uL of the Live/Dead stain wasadded per well, and incubated for 30 minutes at 37° C. The stain wasthen aspirated and washed with PBS 2× prior to imaging. Separatefluorescence images were taken using a Carl Zeiss mercury lamp (N HBO103 Microscope Illuminator) in conjunction with blue (450-490 nm) andgreen (510-560 nm) filters. The color images were merged using WCIFImage J software.

hMSCs were able to attach and spread on all of the silk derivatives, andexhibited similar cell densities and morphologies up to day 5 (FIG. 7a). However, dramatic differences in morphology were observed when thehMSCs reached ˜70% confluence (FIG. 7 b). hMSCs grown on hydrophobicketone azosilk-3 and heptyloxy azosilk-5 silk grew evenly across thesurface and exhibited spindle-shaped morphologies similar to cells grownon TCP. These morphologies are typical of undifferentiated hMSCs, whichtake on a fibroblastic cell shape. Cells grown on unmodified silk orsilks modified with amino, carboxylic acid or sulfonic acid functionalgroups were also spindle-shaped, but they tended to form large star-likeclusters rather than forming a monolayer.

In order to determine if the cell clustering seen on the hydrophilicsubstrates was a result of cell death, cell viability was assessed usinga Live/Dead assay and imaged with a fluorescent microscope.Representative images are shown in FIG. 8 for hMSCs grown on carboxylicacid azosilk-1 and heptyl azosilk-5. All of the cells fluoresce greenindicating that even the clustered cells are still viable. The smallamount of red seen in the image for heptyl azosilk-5 is likely due tothe autofluorescence of silk at this wavelength (Georgakoudi I, Tsai I,Greiner C, Wong Po Foo C, DeFelice J, Kaplan D L. Optics Express 2007;15(3):1043-1053) as the phase contrast images reveal that there are nocells in those regions.

Cell Adhesion via Integrins

After one week of osteogenic stimulation, the cells in each well werelysed in 0.35 mL Buffer RLT (Qiagen) containing 10% mercaptoethanol,followed by shredding in a QlAshedder (Qiagen #79656). RNA was isolatedfrom the cells using an RNeasy Mini Kit (Qiagen #74106). From this RNA,cDNA was synthesized using a High Capacity cDNA Reverse TranscriptionKit (Applied Biosystems #4368814) following the manufacturer'sinstructions. The cDNA samples were analyzed for expression ofα-procollagen I, alkaline phosphatase, osteopontin, bone sialoprotein,and integrin subunits αv, β3, α5 and β1 relative to the GAPDHhousekeeping gene using Assay-on-Demand® Gene Expression kits withTaqMan® Universal PCR Master Mix (ABI #4364340). (Applied Biosystems AoDprobes: Col I #Hs00164004_m1, ALP #Hs00240993_m1, BSP # Hs00173720_m1,OP # Hs00167093_m1, αv #Hs00233808_m1, β3 #Hs00173978_m1, α5#Hs00233743_m1, β1 #Hs00236976_m1, GAPDH #Hs00240993_m1) The data wasanalyzed using the ABI Prism 7000 Sequence Detection Systems software.For each sample, the Ct value was defined as the cycle number at whichthe amplification of each target gene was in the linear range of thereaction. Relative expression levels of each gene were calculated bynormalizing to the Ct value of the housekeeping gene GAPDH (2^(ΔCt),Perkin Elmer User Bulletin #2). Data from three separate cultures ofeach type were averaged.

To evaluate if cell surface integrins played a role in cell adhesion andmorphology on the various silk surfaces, expression of integrin subunitsα_(v), β₃, α₅ and β₁ at day 12 were quantified using real time RT-PCR.As shown in FIG. 9, expression of the αv subunit was highest for cellsgrown on hydrophilic surfaces (contact angle <60°), including unmodifiedsilk, carboxylic acid azosilk-1 and sulfonic acid azosilk-4. Cells grownon amino azosilk-2, heptyl azosilk-5, and TCP had significantly lowerα_(v) expression when compared to cells grown on unmodified silk.Similarly, α₅ expression was lowest for cells grown on TCP and thehydrophobic heptyl azosilk-5. Expression of the β₃ integrin subunit wasalso lower in cells grown on the hydrophobic heptyl azosilk-5 ascompared to cells on unmodified silk, but the difference was not assignificant as for α_(v) expression. No statistical difference inexpression levels of the β₁ integrin subunit was found in cells grown onthe various silk derivatives.

Osteogenic Differentiation

In addition to morphology and growth, hMSCs on the silk derivatives wereevaluated for the ability to differentiate into an osteogenic lineage.At approximately 80% confluence, half of the cells were subjected toosteogenic stimulants and cultured for an additional week. A summary ofosteogenic gene expression using real time RT-PCR analysis is shown inFIG. 10.

Taken as a whole, there were no clear trends in the up or downregulation of osteogenic markers in cells grown on the various azosilkderivatives. Therefore, the modification of silk with azo groups ingeneral does not significantly affect the ability of hMSCs todifferentiate into an osteogenic lineage. However, a few trends wereseen within specific genes that were analyzed. Expression ofα-procollagen I was lower for silk derivatives with either a higher(sulfonic acid azosilk-4) or lower hydrophilicity (ketone azosilk-3 andheptyl azosilk-5) than silk. Osteopontin expression was also lower forcells grown on the more hydrophobic silks. Significant up-regulationwhen compared to unmodified silk was only found in bone sialoproteintranscript expression in cells grown on the carboxylic acid azosilk-1.

SUMMARY

A new method for modifying silk fibroin proteins using diazoniumcoupling chemistry is described herein. Silk is mainly composed ofnon-reactive amino acids leaving few options for functionalization.However, methods of the present invention can be used to install smallmolecules with various functional groups including sulfonic andcarboxylic acids, amines, ketones and alkanes. Silk fibroin is verystable in the basic conditions necessary for this reaction, thus themethods described herein are suitable for modifying tyrosine residues inthe silk protein. In addition, the reaction is rapid, usually requiringless than 20 minutes, and all the necessary reagents are commerciallyavailable.

Fibroin has a high molecular weight of ˜390,000 daltons (Zhou, C Z., etal (2001)) so many standard protein analysis techniques, such as massspectral analysis and gel electrophoresis, are not readily amenable tothis protein. However, the extent of the diazonium coupling with theseaniline derivatives could be followed with UV/vis and ¹H-NMRspectroscopy. It was concluded that the diazonium reaction was efficientusing anilines with electron-withdrawing substituents (1, 3 and 4),where ˜70% of the diazonium salt added resulted in azo formation. Incontrast, only ˜20% of the added diazonium salt formed azo derivativeswhen using anilines with electron-donating substituents (2 and 5).

Incorporation of anilines (1-5) were found to alter the overallhydrophilicity of silk, resulting in hydrophilic (sulfonic acidazosilk-4) and hydrophobic (ketone azosilk-3 and heptyl azosilk-5) silkanalogs. This chemical modification also influenced the fibroin proteinstructure, where incorporation of sulfonic acid groups was found toinhibit β-sheet self-assembly, while addition of low levels ofhydrophobic groups catalyzed β-sheet assembly resulting in rapidhydrogel formation. A new method has recently been reported usingultrasonication to induce rapid silk hydrogel formation (Wang X, Kluge JA, Leisk G G, Kaplan D L. Biomaterials 2008; 29, 8, 1054-1064). Thechemical modification strategy outlined here provides an additionalmethod to reproducibly prepare silk hydrogels. By controlling the levelof tyrosine modification or adjusting the concentration of the silksolution, the gelation time can be tailored to occur anywhere from 5minutes to 2 hours after modification.

The influence of the hydrophilicity of the silk derivatives on hMSCattachment and proliferation of hMSCs were also evaluated. The initialattachment, morphology and proliferation rates were the same for hMSCsgrown on all of the silk derivatives for the first 5 days. However,differences in growth and morphology became apparent after culturing thecells for ˜7 days. hMSCs were able to adhere and rapidly proliferate onall the silk derivatives with a water contact angle <70°, but exhibiteda slower growth rate on the more hydrophobic heptyl azosilk-5. Thesedata are consistent with other observations in the literature that cellsprefer to adhere to and proliferate on surfaces with a moderatehydrophilicity (water contact angle=50-70° (Kim, M S., et al (2007);Grinnel, F. (1978)). Differences in morphology were also seen as thecells reached confluence. Cells on the hydrophilic carboxylic acidazosilk-1, amino azosilk-2, and sulfonic acid azosilk-4 surfaces formedlarge cell clusters, seemingly preferring to adhere to each other overthe substrate. In contrast, hMSCs formed monolayers on the hydrophobicketone azosilk-3 and heptyl azosilk-5.

To determine whether cell surface integrins played a role in theobserved change in cell morphology on hydrophilic (contact angle <70°)vs. hydrophobic (contact angle >70°) silk surfaces, expression of thesubunits of α_(v)β₃ and α₅β₁ integrins were evaluated at transcriptlevels. The α_(v)β₃ integrin is known to adhere to proteins such as bonesialoprotein, fibronectin, fibrinogen, and laminin (Hubbell, J A.(1995)) and plays a role in cell migration. In contrast, the α₅β₁integrin binds to regions within fibronectin (Hubbell, J A. (1995)) andis necessary for forming focal adhesion sites. Expression of α_(v) wasmuch lower in cells grown on the hydrophobic silk derivatives in thepresent study. This result is consistent with the literature, where ishas been shown that cells grown on hydrophobic surfaces express lowerlevels of α_(v) and β₃ (Lim, J Y., et al (2005)). In addition, it hasbeen shown that α_(v)β₃ and α₅β₁ have a much lower binding affinity tohydrophobic monolayers (Keselowsky, B G., et al (2004). Endothelialcells have been shown to increase expression of α_(vβ) ₃ and decreaseexpression of α₅β₁ during wound healing to facilitate cell migration(Gao B, Saba T M, Tsan M F. Am J Physiol Cell Physiol 2002;283(4):C1196-205), and over-expression of α_(v)β₃ also been linked to anincrease in cell motility in cancer cells (Vacca A, Ria R, Presta M,Ribatti D, Iurlaro M, Merchionne F, et al. Exp Hematol 2001;29(8):993-1003). Therefore, the rearrangement of the cells into largeclusters when grown on the hydrophilic surfaces may be due to anincrease in α_(v)β₃expression, which could enhance the migratory abilityof these cells. Conversely, cells grown on the hydrophobic silks and TCPexpress lower levels of α_(v)β₃ which could inhibit motility.

Following stimulation, hMSCs grown on all of the new silk derivativeswere found to express osteogenic markers, demonstrating that theseazosilks can support differentiation. Regardless of the hydrophilicityof the surface on which they were grown, all of the hMSCs expressedsimilar levels of the α₅β₁ integrin which is important for osteogenicdifferentiation (Takeuchi Y, et al. J Biol Chem 1997; 272(46):29309-16;Xiao G, et al. J Biol Chem 1998; 273(49):32988-94; Keselowsky B G, etal. J Biomed Mater Res A 2007; 80(3):700-10). Relative up or downregulation of the specific transcripts associated with osteogenesisvaried slightly between the azosilks, but overall differentiationappeared to be unaffected when compared to the control. Further studiesare underway to evaluate hMSC differentiation in 3-dimensional scaffoldsof these azosilks to ascertain whether derivatives with higher surfacecharge (carboxylic acid azosilk-1 and sulfonic acid azosilk-4) canfacilitate mineralization during osteogenesis (Chen, J., et al (2006);Goissis, G., et al (2003)).

Modification of silk fibroin using diazonium coupling chemistry providesa simple route to controlling protein structure and overallhydrophilicity. When hydrophobic and hydrophilic silk derivatives areused as cell culture scaffolds, cells display different growth rates andmorphologies. However, hMSCs grown on all the silk derivatives are ableto express osteogenic markers when subjected to osteogenic stimuliregardless of the silk hydrophilicity. These data indicate that thisversatile chemistry is useful for studies of silk structure andassembly, while also providing new options for cell cultivation.

The contents of all references cited below, and throughout thisapplication, are incorporated herein by reference.

TABLE 1 Water contact angle values after modification of silk withazosilk 1-5. Silk Derivative Contact Angle Unmodified Silk 58 ± 5°Carboxylic Acid Azosilk-1 60 ± 3° Amino Azosilk-2 56 ± 3° KetoneAzosilk-3 78 ± 6° Sulfonic Acid Azosilk-4 43 ± 5° Heptyl Azosilk-5 84 ±6°

1. A method for modifying a surface, the method comprising the steps of: (a) contacting a diazonium salt with a silk polymer solution to form a modified silk mixture, (b) coating a surface with said modified silk mixture, and (c) transforming said modified silk mixture into an insoluble state.
 2. The method of claim 1, wherein said surface comprises an implantable structure.
 3. The method of claim 1, wherein said surface comprises a tissue culture platform.
 4. The method of claim 1 wherein said contacting comprises mixing said silk polymer and said diazonium salt to form a substantially homogeneous mixture.
 5. The method of claim 1, wherein said silk polymer contains at least one tyrosine residue and said diazonium salt contains at least one chemical moiety.
 6. The method of claim 5, wherein said contacting involves reacting at least one of the chemical moieties of the diazonium salt with the tyrosine residue of the silk polymer.
 7. The method of claim 6, wherein said chemical moiety is selected from the group consisting of a sulfonic acid group, a carboxylic acid group, an amine group, a ketone group, an alkyl group, an alkoxy group, a thiol group, a disulfide group, a nitro group, an aromatic group, an ester group, an amide group, and a hydroxyl group.
 8. The method of claim 1, wherein the insoluble silk polymer is hydrophilic.
 9. The method of claim 1, wherein the insoluble silk polymer is hydrophobic.
 10. The method of claim 1, wherein said silk polymer further comprises a bioactive agent.
 11. The method of claim 10, wherein said bioactive agent is selected from the group consisting of a small molecule, a protein, a polypeptide, and a nucleic acid.
 12. The method of claim 11, wherein said bioactive agent is bonded to said chemical moiety.
 13. The method of claim 10, wherein the bioactive agent is a therapeutic agent selected from the group consisting of anti-infectives, antibiotics, antiviral agents, chemotherapeutic agents, anti-rejection agents, analgesics and analgesic combinations, anti-inflammatory agents, hormones, growth factors, bone morphogenic-like proteins, epidermal growth factor (EGF), fibroblast growth factor, platelet derived growth factor (PDGF), insulin like growth factor (IGF-I and IGF-II), transforming growth factors, vascular endothelial growth factor (VEGF)), anti-angiogenic proteins, anti-thrombotics, anti-metabolics, growth factor inhibitors, growth promoters, anticoagulants, antimitotics, fibrinolytics, sedatives, hypnotics, prostaglandins, radiopharamacuticals, cell attachment mediators, and peptides containing integrin binding domains.
 14. The method of claim 1, wherein said silk polymer further comprises a mineral.
 15. The method of claim 14, wherein said mineral is contacted with said silk polymer, prior to contact with said diazonium salt.
 16. The method of claim 14, wherein said mineral is contacted with said silk polymer, after said contact with said diazonium salt.
 17. The method of claim 14, wherein said mineral is biocompatible with bone or cartilage.
 18. The method of claim 1, wherein said silk polymer solution further comprises a cell.
 19. The method of claim 18, wherein said cell is selected from the group consisting of a stem cell, a primary cell, and a cell line.
 20. The method of claim 19, wherein said stem cell is induced to differentiate.
 21. The method of claim 19, wherein said stem cell is maintained in an undifferentiated state.
 22. A surface modified by the method of claim
 1. 